Inhibition of intestinal Niemann-Pick type C1 Like 1 (NPC1L1) transporter, an essential mediator of intestinal cholesterol absorption, has emerged as an important target for management of hypercholesterolemia. Despite such advances in the therapeutic modalities, reducing plasma cholesterol to low levels in patients with high risk for coronary heart disease remains poor. Therefore, identifying novel mechanisms targeting NPC1L1 is warranted for more effective treatment of hypercholesterolemia. The inhibition of NPC1L1, however, triggers the activation of adaptive cellular pathways such as the Sterol Response Element Binding Protein-2 (SREBP2) that could hinder the efficiency of cholesterol lowering drugs. Suppressing these compensatory mechanisms should also be taken into consideration when designing more effective treatment of hypercholesterolemia. Our efforts are focused at investigating the novel mechanisms involved in the inhibition of NPC1L1 as well as delineating roles of intestinal SREBP2 in body cholesterol homeostasis. Our preliminary data showed that proteasomal and lysosomal-dependent pathways are involved in the normal turnover of NPC1L1 protein. Also, inhibition of MAPK pathway caused a decrease in the cellular level of NPC1L1 by decreasing its protein stability. Our findings showed that DNA methylation is a major epigenetic determinant involved in controlling NPC1L1 expression in intestinal epithelia cells. To understand the role of compensatory pathways, we have recently generated novel transgenic mice with intestine- specific overexpression of constitutively active SREBP2 (designated as ISR2 mice). Our data showed an increase in levels of plasma cholesterol in the LDL and VLDL fractions in ISR2 mice indicating that the activation of intestinal SREBP2 alone is sufficient to increase plasma cholesterol. We hypothesized that inhibition of MAPK pathway decreases NPC1L1 protein stability via proteasomal and/or lysosomal degradation, and that DNA methylation is involved in NPC1L1 gene silencing. We further hypothesize that the increase in intestinal lipid synthesis/absorption and chylomicron production by active intestinal SREBP2 lead to an increase in plasma cholesterol. Our studies are designed to address these hypotheses utilizing both in vitro and in vivo models. Studies designed for Specific Aim 1 will investigate the mechanisms involved in the regulation of NPC1L1 protein stability by the MAPK dependent pathway and determine the structural domains of NPC1L1 involved. Specific Aim 2 will systematically investigate mechanisms of NPC1L1 gene silencing by DNA methylation. Studies designed for Specific Aim 3 will focus on investigating the compensatory mechanisms underlying hypercholesterolemia in mice with intestine specific-overexpression of active SREBP2 (ISR2 mice). Our proposed studies are important to elucidate the critical role of the intestine in the maintenance of cholesterol homeostasis and to unravel potential targets for more effective treatment of hypercholesterolemia.